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Preparation method and application of petroleum degrading enzyme preparation

A technology of petroleum degradation and enzyme preparation, applied in the field of environmental biology, can solve the problems of unreported, unable to meet the requirements of rapid treatment of oil pollution, low speed, etc., and achieve high activity, significant economic and social benefits, and high efficiency. Effect

Active Publication Date: 2014-01-01
ECOLOGY INST SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the rate of degradation of petroleum by the bacterial agent is still low, which cannot meet the requirements of rapid treatment of petroleum pollution.
[0006] However, the application of petroleum degrading enzyme preparations in the remediation and control of petroleum pollution has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the preparation of crude enzyme liquid

[0028] Proceed as follows:

[0029] (1) Refrigerate and centrifuge the Actnetobacter calcoaceticus cultured in LB liquid medium at 10,000rpm for 10min, wash with 0.1M pH6.0PBS buffer three times, and collect the bacteria by centrifugation;

[0030] (2) Resuspend the bacteria obtained in step (1) with 10 times the volume of pH6.0 PBS buffer solution, oscillate and stir well, and perform ultrasonic crushing in an ice bath under the conditions of ultrasonic power of 500W and ultrasonic\interval time of 5s\5s 25min, then centrifuge at 4°C and 10000rpm for 10min, discard the precipitate, and prepare the crude enzyme solution;

[0031] (3) The protein concentration of the crude enzyme solution obtained above was determined by the Coomassie Brilliant Blue method. The specific operation steps are: accurately absorb 0.1 mL of the crude enzyme solution, then add 5 mL of Coomassie Brilliant Blue G250 reagent, shake and mix we...

Embodiment 2

[0033] The preparation of embodiment 2 crude enzyme liquid

[0034] The steps described in Example 1, the difference is that in step (2) resuspend the bacteria with 20 times the volume of PBS buffer solution with a pH of 6.0, oscillate and stir well, and when the ultrasonic power is 400W, the ultrasonic interval time is Under the condition of 5s\5s, perform ultrasonic crushing in an ice bath for 25 minutes, then centrifuge at 4°C and 10,000 rpm for 20 minutes, discard the precipitate, and obtain a crude enzyme solution;

[0035] After testing, the protein concentration of the prepared crude enzyme solution was 2.25mg / mL.

Embodiment 3

[0036] The preparation of embodiment 3 petroleum degrading enzyme preparations and petroleum degradation

[0037] Proceed as follows:

[0038] (1) Take the commercially available diatomite and wash it with distilled water for 3 times, dry it at 80°C, and let it cool down to room temperature naturally to prepare the cleaned diatomite;

[0039] (2) Mix the prepared diatomaceous earth after cleaning with the crude enzyme solution prepared in Example 1 at a ratio of 1:10 (mass / volume, g / ml), shake and fix intermittently at 4°C for 5 hours, and centrifuge , the precipitate was taken, and the petroleum-degrading enzyme preparation was obtained by vacuum freeze-drying; it was stored at 4°C for future use;

[0040] (3) To simulate the experiment of petroleum degrading enzyme preparation in water to degrade petroleum, the operation steps are as follows:

[0041] ①Prepare 50mL of 10g / L petroleum solution in a 250mL Erlenmeyer flask, add 1g of petroleum degrading enzyme preparation, an...

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Abstract

The invention relates to a preparation method and an application of a petroleum degrading enzyme preparation. The preparation method comprises the following preparation steps: performing cell breakage on microorganisms for degrading petroleum to prepare crude enzyme liquid; performing mixed adsorption on the crude enzyme liquid and a carrier; and separating and drying to prepare the petroleum degrading enzyme preparation, wherein the microorganisms for degrading the petroleum are acinetobacter calcoaceticus; the collection number of the strains is CGMCC No.3915; and the collection mechanism is Common Microorganism Center of China General Microbiological Culture Collection Center. The invention also comprises the application of the prepared petroleum degrading enzyme preparation to petroleum degradation. An enzyme system of the microorganisms with a petroleum degrading function is fixed by an adsorbent and then is used for degrading soil polluted by the petroleum, so that the degradation efficiency is obviously improved and is increased by 30 to 50 times compared with the microbial degradation speed, and the stability is improved by 15 to 20 times compared with that of the crude enzyme liquid.

Description

technical field [0001] The invention relates to a preparation method and application of a petroleum-degrading enzyme preparation, belonging to the field of environmental biotechnology. Background technique [0002] Due to the rapid development of the petroleum industry, the environmental pollution caused by oil spills is becoming more and more serious, especially the soil pollution. In my country, nearly 100,000 tons of oil-contaminated soil is produced every year, and the area of ​​oil-contaminated land is about 5 million hectares. [0003] At present, the remediation methods of petroleum-contaminated soil mainly include physical and chemical methods, electrochemical methods, and biological methods. Physicochemical and electrochemical methods have the major defects of high cost, and the nutrient structure of the soil will be changed after restoration, making it no longer suitable for crop planting. However, the bioremediation method has become the oil industry due to its si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C02F3/00B09C1/10
Inventor 王加宁张强高永超张闻郭书海陈贯虹
Owner ECOLOGY INST SHANDONG ACAD OF SCI
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